Studying intact systems with both local precision and global scope is a fundamental unmet goal in biology. For example, in the study of the brain, efforts to determine connectivity of small patches of brain, though pioneering, are challenged by the fact that resulting maps will be nearly impossible to interpret because both the brainwide sources and destinations of traced wiring connections, as well as the activity of corresponding cells during behaviorally significant events, will remain unknown. Conversely molecular, electrophysiological, or imaging methods to record population activity are agnostic with regard to brain-wide wiring patterns of recorded cells, creating an enormous gap in understanding of function. We have now laid foundations for addressing this challenge, by integrating chemical engineering, computational optics, and molecular genetics in an approach termed CLARITY. We will develop the approach in the behaving vertebrate CNS, challenging for speed and complexity, but CLARITY will become applicable across biology as we develop platforms for zebrafish, rodents, and primates. In Aim 1, we bring chemical engineering tools to bear, rapidly transforming scattering and impermeable tissues into intact but transparent and macromolecule-permeable (antibody-compatible) form. In Aim 2, we develop activity-readout technology designed to extract volumetric activity (even in freely-moving mammals) that can then be linked to the global wiring and molecular phenotypes. This transformative technology will allow rapid extraction of systems information (dynamics, history, wiring, and molecular phenotypes) from large and intact biological tissues or organs without disassembly, down to millisecond-scale and cellular resolution. In Aim 3, we directly apply CLARITY to behaving mice and zebrafish, rapidly obtaining brain-wide activity patterns of every cell involved in disease-relevant states of fear an reward. This alone will be a milestone achievement in biology, but furthermore these data will also be linked to full molecular and global wiring information of those cells in the same brains, al publicly accessible for mining/searching. Finally in Aim 4 we design and build online datasets for zebrafish, mouse, and primate to broadly serve the community. Computational infrastructure will address handling and public access to the massive amount of data collected (among the largest datasets in all of biology), including the records of activity in every neuron in vertebrate brains during specific experiences linked to molecular and global wiring information. We are experienced with technology outreach and education, and will leverage this experience to achieve the full transformative mission of this new technology. PUBLIC HEALTH RELEVANCE: Psychiatric disease represents the leading cause of disability both in the U.S. and worldwide, but many major pharmaceutical companies are closing down psychiatry programs; among the reasons cited for this withdrawal is the lack of neural circuit-level understanding of disease-related states such as fear and reward, which impairs identification of targets for treatment. Indeed, it is therefore likely important for human health o observe fine details of symptomatic brains without losing larger-scale circuit perspective. The efforts proposed here, in a new suite of technologies called CLARITY, will directly address this challenge, providing the much-needed across-scales, simultaneously detailed and global perspective, that may lead to fundamental insights into psychiatric disease.